Ultrasound contrast media, contrast agents containing the media and method

ABSTRACT

The invention relates to injectable media for ultrasonic echography in the form of microbubbles or microballoons comprising at least two biocompatible substances A and B (gaseous at the body temperature) forming a mixture which when in suspension with usual surfactants, additives and stabilisers provides useful ultrasound contrast agents. At least one of the components (B) in the mixture is a gas whose molecular weight is greater than 80 daltons and whose solubility in water is below 0.0283 ml per ml of water at standard conditions. The presence of the first component (B) in the contrast medium may vary between 0.5 and 41 volume percent. The other component (A) of the ultrasound contrast media is a gas or a mixture of gases whose molecular weight is below 80 daltons. The second component is present in a proportion of between 59-99.5% by vol., and is preferably chosen from oxygen, air, nitrogen, carbon dioxide or mixtures thereof. Gas mixtures described are found to be very effective as ultrasound contrast media. The invention also comprises a method of making the ultrasound contrast medium, the contrast agent and the ultrasound agent kit.

TECHNICAL FIELD

[0001] The invention relates to contrast media for ultrasonic echographyand injectable ultrasound contrast agents comprising dispersions ofmicroparticles (microbubbles, microballoons or microcapsules) carryingthe contrast media. In addition to microparticles the contrast agentscomprise a physiologically acceptable aqueous carrier liquids whichincludes surfactants, additives and stabilisers. The invention alsoconcerns methods of making the ultrasound contrast media and contrastagents and methods of using the same.

BACKGROUND ART

[0002] Recognition of the utility of injectable suspensions of gasmicroparticles as useful ultrasound contrast agents for diagnosticpurposes has triggered considerable research and development towardsimproved dispersions of gas filled microballoons or microbubbles withhigher stability, better resistance to pressure variations, goodechogenicity, ease of manufacture, field use and storage. Many proposalsfor ultrasound contrast agents with such suspensions have been made. Forexample, aqueous suspensions usable as imaging agents in ultrasonicechography are disclosed in WO-A-91/15244 (Schneider et. al.),WO-A-92/11873 (Beller et. al.) or EP-A-0 077 752 (Schering).

[0003] WO-A-91/15244 (Schneider et. al.) discloses microbubblesuspensions containing film forming surfactants in laminar and/orlamellar form and, optionally, hydrophilic stabilizers. The suspensionsare obtained by exposing the laminarized surfactants to air or a gasprior to or after admixing with an aqueous phase. Conversion of filmforming surfactants into lamellar form is carried out according tovarious techniques including high pressure homogenisation or sonicationunder acoustic or ultrasonic frequencies. The reported concentration ofthe microbubbles in these suspensions is between 10⁸ and 10⁹ bubbles/ml.The suspensions disclosed exhibit a fairly high stability duringstorage.

[0004] In WO-A-94/09829 (Schneider et. al.) it is shown thatconcentrations of the laminar and/or lamellar phospholipids used in thepreparations of very stable aqueous suspensions may be as low as tocorrespond to a single monomolecular layer of the phospholipid aroundthe microbubbles in the suspension. Stable, low phospholipid content(down to a few μg/ml) suspensions have been stored for prolonged periodswithout significant loss of microbubble count or echogenicity.

[0005] A method of imparting stability against pressure variations tosuspensions of microbubbles or microballoons used as ultrasound contrastagents is disclosed in EP-A-0 554 213 (Schneider et al.). There, it hasbeen shown that a significant enhancement of the stability of themicrobubbles against collapse due to pressure variations upon injectionmay be achieves if commonly used air, nitrogen or other soluble gasesare at least partially replaced by gases whose in water expressed inlitres of gas by litre of water under standard conditions divided by thesquare root of the molecular weight in daltons does not exceed 0.003.Gases disclosed which satisfy the above criteria are for example, SeF₆,SF₆, CF₄, C₂F₆, C₂F₈, C₄F₁₀, etc. These gases have been found to producelong lasting and in vivo very stable microballoons which in turn providehigh quality echographic images.

[0006] WO-A-92/17212 and WO-A-92/17213 (Klaveness et al.) discloseultrasound contrast agents comprising microballoons having an envelopemade of non-proteinaceous crosslinked or polymerised amphiphilicsubstances (e.g. phospholipids) and crosslinked proteins (e.g. albumin).Microballoons are encapsulating gases such as air, oxygen, hydrogen,nitrogen, helium, argon, CH₄, SF₆ or gas precursors such as sodium orammonium bicarbonate.

[0007] WO-A-93/06869 (Mallinckrodt Medical Inc.) discloses a method ofultrasound imaging of a warm blooded animal in which a pharmaceuticallyacceptable gas or a mixture of gases is administered to the animal andthe animal is scanned with an ultrasound probe. The gases or gasmixtures are administered by inhalation as apparently upon inhalation ofthe mixture for a few minutes, microbubbles will form in the bloodstream of a warm blooded animals and the echographic image of tissuewill change. The gases and gas mixtures disclosed include oxygen,nitrous oxide, C₂H₆, SF₆, xenon, perfluorocarbons, etc. Useful gases andgas mixtures are those which tend to form larger bubbles in the bloodand may be typified by xenon and nitrous oxide and other weakly activegeneral anesthetics such as sulfur hexafluoride. Illustrated mixturescontain either 20% of oxygen, 60-80% of sulfur hexafluoride, and/or 20%of nitrogen, xenon, nitrous oxide or ethylene or 20% of oxygen, 20% ofnitrogen and 60% of xenon or nitrous oxide. The method is based oncomparison of ultrasonic signals obtained during two different scans.The first, prior to inhalation of the gas mixture and the second, sometime after inhalation.

[0008] An interesting concept has been disclosed in WO-A-93/05819(Quay). The document discloses emulsions of liquid dodecafluoropentaneor decafluorobutane and sorbitol in water which upon injection formgaseous microbubbles which resist pressure variations and provide a goodechogenic signal. The substances in the emulsions, although liquid atambient temperature, are highly volatile and easily vaporize at bodytemperature and form gaseous dispersions in a carrier liquid containingadditives and stabilisers such as sorbitol. Upon injection, the dropletsof the highly volatile substance rapidly disaggregate and generate afair amount of very persistent microbubbles. The microbubbles which onlycontain the chosen substance e.g. dodecafluoropentane in pure form atexclusion of air or any other gas are stabilised by stabilising agents,e.g. sorbitol, Tween® 20 and soybean oil which are present in theemulsion carrier liquid. By generalisation, Quay found that theforegoing technique was applicable to a number of other non-liquid(gaseous) chemical substances which were brought into use via a criteriadefined as a relationship between volume density, solubility anddiffusivity (coefficient Q). The document claims that any biocompatiblegas whose coefficient Q is greater than 5 is potentially useful as anechographic agent, and a list of about 180 gases/liquids which satisfythe criteria is presented. It follows from the document that to achievethe desired properties, contrast agents are to be made with substanceswhose coefficient Q must be greater than 5. The criteria defined isQ=4.0×10⁻⁷×ρ/C_(s) D where ρ is density of the gas, D is diffusivity ofthe gas in solution and C_(s) is the water solubility of the gas, andthis has been developed using a simple model in which diffusivities andsolubilities of gases in water are used as the approximation closest toreality. Contrast agents obtained from pure i.e. non-admixed, substanceschosen according to the above criteria have shown encouraging results.Tested on experimental dogs, the contrast agents have been reported tofurnish promising results in the echography of the myocardium afterperipheral venous injections (see Beppu S. et al. in Proceedings from66^(th) Scientific Session of the American Heart Association, Atlanta,October 1993). Depending on the dose, injections of 2.2% emulsion ofdodecafluoropentane have been found to provide a mean opacificationduring up to 85 minutes. However, with doses at which opacification ofthe left heart was homogeneous, there was observed a decrease in oxygensaturation of arterial blood and an increase of pulmonic arterialsystolic pressure were observed.

[0009] Many of the prior art compositions have merit and many are underintensive clinical tests. Many are at various stages of development.From various reports it however appears that, to date, only a very smallnumber of contrast agents is capable of exploiting the full range ofdiagnostic possibilities basically provided by ultrasound echography.Indeed, only a few contrast agents are really useful and help themedical profession to profit from the diagnostic technique which,otherwise, represents one of the best non-invasive methods for analysingorgans in the human body. Not many agents allow exploitation of the fullpotential of the ultrasound concept and this hampers wider use of thetechnique and/or of the imaging agents. Experimentation with the knownechographic agents has shown that some provide insufficient backscatterto ensure good intensity and contrast or provide useful images only incertain percentage of the population which limits their utility as adiagnostic tool of general use. Others, because of poor resistance topressure variations, are too short lived to allow meaningfulmeasurements or useful images. Typically, contrast agents whosemicrobubbles or microballoons are filled with gases of high solubilityin water poorly resist pressure variations. Suspensions of microballoonswhose envelope is made from rigid materials are also ineffective as theydo not resonate sufficiently in response to the acoustic waves.Noteworthy contrast agents which have a high resistance to pressurevariations are those using gases with low solubilities in the aqueouscarrier. The direct consequence of low solubility is low rate ofresorption and slow elimination from the body. Imaging agents made fromsuch very insoluble gases remain in the blood circulation for prolongedperiods causing relapse or recirculation of the gas microbubbles whichcauses interference with images produced during the initial stages ofthe test. Such contrast agents are generally useful for imaging the leftheart but because of slow resorption or elimination, they cannot be usedeffectively for perfusion measurements. Perfusion measurements areusually carried out by integration of the echographic response curve,this being a typically Gaussian function, appearing after a “singlepass” of the imaging agent. Relapse or recirculation after the “singlepass” is therefore undesirable, as the repetition would superpose andimpair the final result. It is therefore generally admitted that thepersistence over a certain period of the microbubbles or microballoonsendowed with high pressure resistance is more disturbing than helpful.Echographic contrast agents with very persistent microbubbles are usefulonly for certain studies, e.g. vascular Doppler investigations. Agentsused for imaging of the left heart and myocardium should provide clearimages and should have good resistance to pressure variation but shouldnot be overlasting and should not disturb images created immediatelyupon injection. Recirculation is not a desirable feature of agents whoseintended use is to cover a range of applications and clear imaging.Obviously, it is highly desirable to modulate the pressure resistance orpersistence of the contrast agent after injection, i.e. to usesuspensions of bubbles (or microballoons) designed with sufficientpressure resistance but with controlled life-time in the circulation.This demand is fulfilled by the invention below.

SUMMARY OF THE INVENTION

[0010] Briefly summarised, the invention relates to an injectableultrasound contrast medium in the form of microbubbles or microballoonscomprising at least two biocompatible, at the body temperature gaseous,substances A and B forming a mixture which when in suspension with usualsurfactants, additives and stabilisers provides useful ultrasoundcontrast agents. At least one of the components (B) in the mixture is agas whose molecular weight is above 80 daltons and whose solubility inwater is below 0.0283 ml of gas per ml of water under standardconditions. Through out this document gas solubilities referred tocorrespond to the Bunsen coefficients and the molecular weights above 80daltons are considered as relatively high, while the molecular weightsbelow 80 daltons are considered as relatively low. The mixtures of theinvention therefore may be defined as mixtures of in which the majorportion of the mixture is comprised of “a relatively low” molecularweight gas or gases, while the minor portion of the mixture is comprisedof “a relatively high” molecular weight gas or gas mixture. The quantityof this “minor” or activating component (B) in the contrast medium ispractically always between 0.5 and 41 volume percent. The othercomponent (A) of the ultrasound contrast media may be a gas or a mixtureof gases whose solubility in water is above that of nitrogen (0.0144ml/ml of water under standard conditions) and whose quantity in themixture is practically always in a proportion of between 59-99.5% byvol. This “major” or dominating component is preferably a gas or gaseswhose molecular weights are relatively low, usually below 80 daltons,and is chosen from gases such as oxygen, air, nitrogen, carbon dioxideor mixtures thereof.

[0011] In the ultrasound contrast medium of the invention the gas whosemolecular weight is above 80 daltons may be a mixture of gases ormixture of substances which are gaseous at body temperature but which,at ambient temperatures, may be in the liquid state. Such gaseous orliquid substances may be useful in the contrast media of the inventionas long as the molecular weight of each such substance is greater than80 daltons and the solubility in water of each substance is below 0.0283ml of gas per ml of water under standard conditions.

[0012] When filled with the contrast media of the invention anddispersed in an aqueous carrier containing usual surfactants, additivesand stabilisers, the microbubbles formed provide an injectable contrastagent for ultrasonic imaging, of controlled resistance to pressurevariations and modulated persistence after injection. In addition to themicrobubbles, the contrast agent of the invention will containsurfactants stabilising the microbubble evanescent gas/liquid envelope,and optionally, hydrophilic agents and other additives. The additivesmay include block copolymers of polyoxypropylene and polyoxyethylene(poloxamers), polyoxyethylene-sorbitans, sorbitol, glycerol-polyalkylenestearate, glycerolpolyoxlethylene ricinoleate, homo- and copolymers ofpolyalkylene glycols, soybean-oil as well as hydrogenated derivatives,ethers and esters of sucrose or other carbohydrates with fatty acids,fatty alcohols, glycerides of soya-oil, dextran, sucrose andcarbohydrates. Surfactants may be film forming and non-film forming andmay include polymerizable amphiphilic compounds of the type oflinoleyl-lecithins or polyethylene dodecanoate. Preferably, thesurfactants comprise one or more film forming surfactants in lamellar orlaminar form selected between phosphatidic acid, phosphatidylcholine,phosphatidyl-ethanolamine, phosphatidylserine, phosphatidylglycerol,phosphatidyl-inositol, cardiolipin, sphingomyelin and mixtures thereof.

[0013] The invention also comprises a method of making the ultrasoundcontrast agents by suspending in a physiologically acceptable carriercontaining usual surfactants and stabilisers, gas filled microbubbles ormicroballoons comprising a mixture of gases at least one of which is agas whose minimum effective amount in the mixture may be determinedaccording to the expression:

B _(c)%=K/e ^(bMwt) +C

[0014] in which B_(c)% (by vol.) is the total quantity of the componentB in the mixture, K, C & b are constants with values of 140, −10.8 and0.012 respectively, M_(wt) represents the molecular weight of thecomponent B exceeding 80. The contrast agents made according to thepresent method comprise suspensions of microbubbles or microballoonswith excellent resistance to pressure variations and a controlledresorption rate.

[0015] The invention also includes a kit comprising a dry formulationwhich is usually stored under a mixture of gases and/or liquids that areconverted into gases at body temperature. When dispersed in aphysiologically acceptable carrier liquid, the dry formulation with themixture of gases and/or liquids produces the ultrasound contrast agentof the invention. A method of storage of the dry lyophilised formulationin the presence of the ultrasound contrast media is also disclosed.

[0016] The invention further comprises a method of making contrastagents with microbubbles containing the ultrasound contrast media, aswell as their use in imaging of organs in human or animal body.

BRIEF DESCRIPTION OF THE DRAWINGS

[0017]FIG. 1 is a schematic presentation of an ultrasound contrastmedium according to the invention.

[0018]FIG. 2 is schematic diagram of the critical pressure (Pc) of thecontrast medium as a function of the quantity of a chosen gas in themixture.

[0019]FIG. 3 represents a diagram of the critical pressure (Pc) of acontrast medium made with octafluorocyclobutane (C₄F₈) anddodecafluoropentane (C₅F₁₂) as a function of quantity of gas in themixture.

[0020]FIG. 4 is a diagram of the minimum amount of a gas in the mixtureas a function of the molecular weight.

[0021]FIG. 5 is a graphic representation of the in vivo echographicresponses obtained as a function of time in the left ventricle of aminipig after intravenous injection of contrast media containing variousconcentrations of SF₆.

[0022]FIG. 6 represents a diagram of in vivo echographic responseobtained as a function of time with contrast media containing variousconcentrations of C₄F₈.

DETAILED DESCRIPTION OF THE INVENTION

[0023] This invention is based on the unexpected finding that anultrasound contrast medium comprising bubbles filled with a mixture ofat least two biocompatible gaseous or at body temperature gaseoussubstances A (major or a relatively low molecular weight) and B(activating or a relatively high molecular weight), will provide, insuspension with usual surfactants, additives and stabilisers, injectableultrasound contrast agents that combine desirable resistance to pressureand a shorter life time in the circulation, both of these parametersbeing controllable at will. As long as at least one of the (activating)substances or components in the mixture with molecular weight greaterthan 80 daltons (relatively high molecular weight) is present in certainminimal proportion and as long as its solubility in water is below0.0283 ml of gas per ml of water at standard conditions, the ultrasoundcontrast medium will provide echographic properties as good as thatobtained when using the pure substances alone. By “activating” it ismeant the substance or component which imparts its physical propertiesto the other components in the mixture rendering the mixture, in termsof echogenicity and resistance to pressure variations, behave the sameor almost the same as the substance or component alone (in pure form).The quantity of the first, activating or high molecular weight,component in the contrast medium in most cases vary from as low as 0.5volume percent (for substances with high molecular weight and lowsolubility in water) to 41 volume percent. The experiments have shownthat substances with the molecular weight below 80 daltons (“lowmolecular weight”) are not suitable as the activating components andthat the upper limit of the molecular weight is difficult to establishas all compounds tested were effective as long as their molecular weightwas relatively high i.e. above 80. Thus compounds with the molecularweight of about 240 daltons such as decafluorobutane or 290 daltons suchas perfluoropentane have been found as effective activating component.Also there are indications that substances such as 1,2,3-nonadecanetricarboxylic acid, 2-hydroxy-trimethylester with the molecular weightsightly over 500 daltons may also be used as an activating, highmolecular weight, component. The other “major” component iscorrespondingly present in an amount of 59 to 99.5% by volume and may bea gas or gases whose solubility in water is greater than that ofnitrogen (0.0144 ml/ml of water under standard conditions). The secondcomponent is preferably oxygen, air, nitrogen, carbon dioxide ormixtures thereof and more preferably oxygen or air. However, for thecomponent A, other less common gases like argon, xenon, krypton, CHClF₂or nitrous oxide may also be used. Some of these less common gases mayhave molecular weights higher than that of O₂, N₂, air, CO₂, etc., forinstance above 80 daltons but, in this case, their solubility in waterwill exceed that of the gases of cathegory B i.e. will be above 0.0283ml/ml of water.

[0024] It was quite unexpected to find that suspending in an aqueouscarrier a mixture formed of as little as 0.5% by volume of a substancesuch as dodecafluoropentane, or 0.8% by volume of decafluorobutane inadmixture with air will produce microbubbles giving excellentechographic images in vivo and resistance to pressure variations. Thisis particularly surprising since it was heretofore considered necessarythat in order to obtain good echographic images of the left heart andthe myocardium, these substances, and for that matter a number ofothers, be used at 100% concentrations, i.e. in pure form (without air).Experiments with mixtures containing different amounts of these, lowwater solubility, substances and air have shown that the echographicimages are as good as those obtained under similar conditions usingechographic agents made with only pure substances.

[0025] Early studies have shown that rapid elimination of airmicrobubbles in the circulation takes place because this otherwisephysiologically preferred gas is quickly resorbed by dilution and thatevanescence of the microbubbles may be reduced through the use ofvarious surfactants, additives and stabilisers. In the early days ofdevelopment, as a cure to the evanescence problem, microballoons ormicrovesicles with a material wall have also been proposed.Microvesicles with walls made from natural or synthetic polymers such aslipid bilayers (liposomes) or denaturated proteins like albumin filledwith air or CO₂ have been proposed. The poor resistance to pressurevariations and the consequent loss of echogenicity of the older contrastagents has inspired a search for gaseous particles with greaterresistance to the pressure variations occurring in the blood stream.Hence, filler gases such as sulfur hexafluoride of more recentlydodecafluoropentane have been proposed. Experimentation with these gaseshave indicated that upon injection, the suspensions of microbubbles madewith these gases taken alone are indeed very resistant to collapse inthe blood circulation. As a result of these initial findings, close to200 different gases have been identified as potentially useful formaking ultrasound contrast agents. It has thus been unexpectedly foundthat by mixing oxygen or air with some of these gases resistant topressure one may obtain ultrasound agents which will havephysiologically better tolerance and/or shorter resorption half-lifethan pure sulfur hexafluoride or dodecafluoropentane, still retainingthe good pressure resistance of these gases when taken alone. It ispostulated that such surprising behaviour of the ultrasound medium ofthe invention comes from the fact that in the microbubbles containingthe gas mixtures diffusion of air into surrounding liquid is slowed bythe presence of the large molecules of gas or gases whose solubilitiesin water are about the same or lower than that of air or oxygen.Although the reasons for this surprising behaviour are yet unexplained,it can be postulated that the molecules of the high molecular weightgas, even though in very minor amount, do actually “plug the holes” inthe microbubbles boundary and thus prevent escape of the low molecularweight gas by transmembrane diffusion. A graphical presentation of thismodel is shown in the FIG. 1 where the microbubble containing air (1)admixed with a gas whose molecular weight is above 80 daltons (2) issuspended in an aqueous medium (3). The evanescent outer layer (4)stabilised by a surfactant (e.g. phospholipid) keeps the gas mixturewithin contained volume defining the microbubble. The activating orminority gas B being uniformly dispersed through out the microbubblevolume will have a slower diffusion and ultimatelly will block the poresof, in the aqueous solution spontaneously formed surfactantmembrane-like envelope, thus preventing rapid departure of the smallerand typically more soluble majority component A. On the other hand, theactivating or minor component gas (B) exhibit greater affinity for thelipophilic part of the surfactant used for stabilisation of theevanescent envelope than oxygen or air. Thus according to anotherhypothesis these gases tend to concentrate in the vicinity of themembrane preventing or slowing diffusion of the smaller gas(es) acrossthe membrane. Be that as it may, the experimental data gathered suggestthat for preparation of echographic media of the invention, the requiredamount of the activating gas in the mixture is that which corresponds toblocking the porosity of the given membrane material or to the amountrequired for a monomolecular layer formed on the inner wall of themicrobubbles. Therefore, the minimum amount required is that which isneeded to block the pores or cover the inner wall of the membrane toprevent escape and resorption of the low molecular weight component.

[0026] It is also believed that the superior properties of theultrasound contrast medium of the invention comes form the combined useof nitrogen, carbon dioxide, oxygen or air (essentially anoxygen/nitrogen mixture) with other gases. Functionally, thesebiologically and physiologically compatible gases provide importantcharacteristics of the media in question thus ensuring theiradvantageous properties. Although, the ultrasound contrast media of theinvention may be made with a number of other gases serving as themajority or component A, oxygen and air are preferred. In the context ofthis document air is treated as a “one component” gas.

[0027] According to the invention, ultrasound contrast media with highresistance to pressure variations combined with relatively rapidresorption, i.e. clearance in the body can be obtained when using a gasor gases whose molecular weights is/are above 80 daltons in admixturewith gas or gases whose solubilites in water are greater than 0.0144ml/ml of water and molecular weight(s) is/are usually below 80 daltons.Gases such as oxygen or air mixed with substances which are gases at thebody temperature but which at the ambient temperatures may be in theliquid state will produce echographic media that will possess alladvantages of the gases in the mixture. In other words these mixtureswhen injected as suspensions of microbubbles will provide clear andcrisp images with sharp contrasts (typical for microbubbles with goodresistance to pressure variations) and at the same time will be resorbedsubstantially as easily as if filled with air or oxygen only. Thus bycombining air, nitrogen, carbon dioxide or oxygen with a certaincontrolled amount of any of the known biocompatible high molecularweight substances which at the body temperature are gases, ultrasoundcontrast media with important and totally unexpected advantages areobtained. As explained above, these media provide the best of eachcomponents i.e. a good resistance to pressure variations from one and arelatively rapid resorption from the other and at the same timeeliminating respective disadvantages of each component taken alone inthe media. This is particularly surprising as one would have expectedproperties averaging those of the components taken separately.

[0028] As long as the molecular weight of such biocompatible substances(B) is greater than 80 daltons and their solubility in water is below0.0283 ml of gas per ml of water under standard conditions, suchsubstances in the gaseous or liquid state are useful for the contrastmedia of the invention. Although in conjunction with suitablesurfactants and stabilisers, gases like sulfur hexafluoride,tetrafluoromethane, chlorotrifluoromethane, dichlorodifluoro-methane,bromotrifluoromethane, bromochlorodifluoromethane,dibromo-difluorornethane dichlorotetrafluoroethane,chloropentafluoroethane, hexafluoroethane, hexafluoropropylene,octafluoropropane, hexafluoro-butadiene, octafluoro-2-butene,octafluorocyclobutane, decafluorobutane, perfluorocyclopentane,dodecafluoropentane and more preferably sulfur hexafluoride and/oroctafluorocyclobutane, may be used in category B, the media of theinvention preferably contains as gas B a gas selected from sulfurhexafluoride, tetrafluoromethane, hexafluoroethane,hexafluoro-propylene, octafluoropropane, hexafluorobutadiene,octafluoro-2-butene, octafluorocyclobutane, decafluorobutane,perfluorocyclopentane, dodecafluoropentane and more preferably sulfurhexafluoride and/or octafluorocyclobutane.

[0029] Another unexpected and surprising feature of the invention is thefact that when the criteria of WO 93/05819 are applied to the media ofthe present invention the Q coefficient obtained with the present gasmixtures is below 5. This is astounding since, according to WO 93/05819media with Q coefficients below 5 are to be excluded from gases suitablefor preparing useful ultrasound contrast media. Nevertheless, it hasbeen found that the uniform gas mixtures of the present inventionalthough having a Q coefficient well below 5, still provide contrastagents useful for ultrasound imaging.

[0030] When filled with the contrast media of the invention anddispersed in an aqueous carrier containing usual surfactants, additivesand stabilisers, the microbubbles formed provide a useful contrast agentfor ultrasonic imaging. In addition to the microbubbles, the contrastagent of the invention will contain surfactants additives andstabilizers. Surfactants which may include one or more film formingsurfactants in lamellar or laminar form are used to stabilize themicrobubble evanescent gas/liquid envelope. Hydrating agents and/orhydrophilic stabilizer compounds such as polyethylene glycol,carbohydrates such as lactose or sucrose, dextran, starch, and otherpolysaccharides or other conventional additives like polyoxypropyleneglycol and polyoxyethylene glycol; ethers of fatty alcohols withpolyoxyalkylene glycols; esters of fatty acids with poloxyalkylatedsorbitan; soaps; glycerol-polyalkylene stearate;glycerol-polyoxyethylene ricinoleate; homo- and copolymers ofpolyalkylene glycols; polyethoxylated soya-oil and castor oil as well ashydrogenated derivatives; ethers and esters of sucrose or othercarbohydrates with fatty acids, fatty alcohols, these being optionallypolyoxyakylated; mono-, di- and triglycerides of saturated orunsaturated fatty acids; glycerides of soya-oil and sucrose may also beused. Surfactants may be film forming and non-film forming and mayinclude polymerizable amphiphilic compounds of the type oflinoleyl-lecithins or polyethylene dodecanoate. Preferably, thesurfactants are film forming and more preferably are phospholipidsselected from phosphatidic acid, phosphatidylcholine,phosphatidylethanolamine, phosphatidylserine, phosphatidylglycerolphosphatidylinositol, cardiolipin, sphingomyelin and mixtures thereof.

[0031] It is understood that the invention is not limited to thecontrast agents in which only microbubbles are used as carriers of theultrasound contrast media of the invention. Any suitable particle filledwith the ultrasound contrast medium e.g. liposomes or microballoonshaving an envelope produced from synthetic or natural polymers orproteins may conveniently be used. Thus it has been established thatmicroballoons prepared with albumin, or liposome vesicles or iodipamideethyl ester porous particles when filled with the ultrasound contrastmedia of the invention, provide good echographic contrast agents.Suspensions in which the microbubbles were stabilised with sorbitol ornon-ionic surfactants such as polyoxyethylene/polyoxypropylenecopolymers (commercially known as Pluronic®) have demonstrated equallygood imaging capability when compared to that of the originalformulations made with the pure substances taken alone. It is therefore,believed that the invention offers a more generalised concept ofultrasound media and offers better insight into the problems ofultrasound imaging as well as better control of contrast agentproperties. The media and contrast agents containing the media of theinvention are, therefore, considered as products which take thetechnique one step further in its development.

[0032] The invention also comprises a method of making the ultrasoundcontrast agent, in which a gas mixture of at least two components issuspended in a physiologically acceptable aqueous carrier liquidcontaining usual surfactants and stabilisers so as to form gas filledmicrobubbles or microballoons, characterised in that the minimumeffective proportion of at least one gas component (B) in said mixtureof gases is determined according to the criteria

B _(c)%=K/e ^(bMwt) +C

[0033] in which B_(c)% (by vol.) is the total quantity of the componentB in the mixture, K & C are constants with values of 140 and −10.8respectively, M_(wt) represents the molecular weight of the component Bexceeding 80 and b is quantity that is a complex function of operatingtemperature and thickness of the membrane (a lipid film) that stabilizesthe microbubbles; however, since the body temperature is substantiallyconstant and the stabilizer film structure substantially independent oflipid concentration, the value of b keeps in the interval 0.011-0.012and it may be considered as constant. The contrast agents made accordingto the method comprise suspensions of microbubbles or microballoons withexcellent resistance to pressure variations and a relatively rapidresorption. Both of the properties are controlled to the extent thatpractically custom-tailored echographic agents are now possible. Withthe above criteria it is possible to produce an agent with desiredcharacteristics starting from any available non-toxic (“of the shelf”)substance which at body temperature is gas and which has the molecularweight and solubility in water as explained above.

[0034] The invention also includes a dry formulation comprisingsurfactants, additives and stabilisers stored under a mixture ofsubstances which at the body temperature are gases at least one of whichis a gas whose molecular weight is greater than 80 daltons and whosesolubility in water is below 0.0283 ml per ml of water under standardconditions. Prior to injection the formulation comprising lyophilisedfilm forming surfactants and optionally, hydrating agents likepolyethylene glycol or other conventional hydrophilic substances, isadmixed with a physiologically acceptable carrier liquid to produce theultrasound contrast agent of the invention. The film forming surfactantis, preferably, a phospholipid selected from phosphatidic acid,phosphatidylcholine, phosphatidylethanolamine, phosphaltidylserine,phosphatidylglycerol phosphatidylinositol, cardiolipin, sphingomyelinand mixtures thereof.

[0035] In a variant, stabilisation of the microbubble evanescentgas/liquid envelope may be secured by non-ionic surfactants such ascopolymers of polyoxyethylene and polyoxypropylene in combination with afilm forming surfactant such as dipalmitoylphosphatidylglycerol. Asbefore the aqueous liquid carrier may further contain hydrophilicadditives such as glycerol, PEG, sorbitol, etc. Furthermore, usefulagents of the invention may be prepared with saline solutions containingTween® 20, sorbitol, soybean oil, and optionally other additives.

[0036] Also disclosed is a two-component kit comprising as the firstcomponent a dry formulation of surfactants, additives and stabilisersstored under a mixture of gases and as the second component aphysiologically acceptable carrier liquid which when brought in contactwith the first component provides an ultrasound contrast media. The kitmay include a system of two separate vials, each containing one of thecomponents, which are interconnected so that the components may beconveniently brought together prior to use of the contrast agent.Clearly, the vial containing the dry formulation will at the same timecontain the ultrasound medium of the invention. Conveniently, the kitmay be in the form of a pre-filled two compartment syringe and mayfurther include means for connecting a needle on one of its ends.

[0037] The invention further comprises a method of making contrastagents with microbubbles containing the ultrasound contrast media, aswell as their use in imaging of organs in human or animal body.

[0038] When used for imaging of organs in human or animal body theultrasound contrast medium of the invention is administered to thepatient in the form of an aqueous suspension in the above describedphysiologically acceptable carrier liquid and the patient is scannedwith an ultrasound probe whereby an image of the organ or the part ofthe body imaged is produced.

[0039] The following examples further illustrate the invention:

EXAMPLE 1

[0040] Multilamellar vesicles (MLVs) were prepared by dissolving 120 mgof diarachidoylphosphatidylcholine (DAPC, from Avanti Polar Lipids) and5 mg of dipalmitoylphosphatidic acid (DPPA acid form, from Avanti PolarLipids) in 25 ml of hexane/ethanol (8/2, v/v) then evaporating thesolvents to dryness in a round-bottomed flask using a rotary evaporator.The residual lipid film was dried in a vacuum dessicator and afteraddition of water (5 ml), the mixture was incubated at 90° C. for 30minutes under agitation. The resulting solution was extruded at 85° C.through a 0.8 μm polycarbonate filter (Nuclepore®). This preparation wasadded to 45 ml of a 167 mg/ml solution of dextran 10000 MW (Fluka) inwater. The solution was thoroughly mixed, transferred in a 500 mlround-bottom flask, frozen at −45° C. and lyophilised under 13.33 Nt/m²(0.1 Torr). Complete sublimation of the ice was obtained overnight.Aliquots (100 mg) of the resulting lyophilisate were introduced in 20 mlglass vials. The vials were closed with rubber stoppers and the airremoved from vials using vacuum. Mixtures of air with various amounts ofsulfur hexafluoride were introduced into the vials via a needle throughthe stopper.

[0041] Bubble suspensions were obtained by injecting in each vial 10 mlof a 3% glycerol solution in water followed by vigorous mixing. Theresulting microbubble suspensions were counted using a hemacytometer.The mean bubble size was 2.0 μm. In vitro measurements (as defined inEP-A-0 554 213) of the critical pressure (Pc), echogenicity (i.e.backscatter coefficient) and the bubble count for various samples wereperformed (see Table 1).

[0042] As it may be seen from the results, the microbubbles containing100% air (sample A) have a low resistance to pressure. However, withonly 5% SF₆, the resistance to pressure increases considerably (sampleB). With 25% SF₆ TABLE 1 Concen- air SF₆ Q Pc Echogenicity trationSample % vol % vol coeff. mmHg 1/(cm · sr) × 100 (bubbles/ml) A 100 01.0 43 1.6 1.5 × 10⁸ B 95 5 1.3 68 2.1 1.4 × 10⁸ C 90 10 1.6 85 2.4 1.5× 10⁸ D 75 25 3.1 101 2.3 1.4 × 10⁸ E 65 35 4.7 106 2.4 1.5 × 10⁸ F 5941 5.8 108 2.4 1.6 × 10⁸ G 0 100 722.3 115 2.3 1.5 × 10⁸

[0043] the resistance to pressure is almost identical to that of 100%SF₆. On the other hand, the bubble concentrations, the mean bubble sizesand the backscatter coefficients are almost independent of thepercentage of SF₆.

[0044] The resulting suspensions were injected intravenously intominipigs (Pitman Moore) at a dose of 0.5 ml per 10 kg and the images ofthe left ventricular cavity were recorded on a video recorder. In vivoechographic measurements were performed using an Acuson XP128 ultrasoundsystem (Acuson Corp. USA) and a 7 MHz sector tranducer. The intensity ofthe contrast was measured by video densitometry using an image analyser(Dextra Inc.). FIG. 5 shows the video densitometric recordings in theleft heart of a minipig. Again a considerable difference is observedbetween the 100% air case (sample A) and the 95% air case (sample B). Inparticular, with 5% SF₆ the maximum intensity is already almost achievedand the half life in circulation shows also a very rapid increase. With10% SF₆, there is no additional increase in intensity but only aprolongation of the half-life. From the example, it follows that usingmore than 10% to 25% SF₆ in the gas mixture provides no real benefit. Itis interesting to note that the values of the Q coefficient obtained forthe mixtures used were well below the critial value of 5 stipulated byWO-A-93/05819.

EXAMPLE 2

[0045] Aliquots (25 mg) of the PEG/DAPC/DPPA lyophilisate obtained asdescribed in Example 1 (using PEG 4000 instead of dextran 10,000) wereintroduced in 10 ml glass vials. Tedlar® sampling bags were filled withair and octafluorocyclobutane (C₄F₈). Known volumes were withdrawn fromthe bags by syringes and the contents thereof were mixed via a three waystopcock system. Selected gas mixtures were then introduced into theglass vials (previously evacuated). The lyophilisates were thensuspended in 2.5 ml saline (0.9% NaCl). The results presented below showthe resistance to pressure, the bubble concentration and the backscattercoefficient of the suspensions. In the case of 100% C₄F₈ the resistanceto pressure reached to 225 mm Hg (compared to 43 mm Hg in the case ofair). Again a considerable increase in pressure resistance was alreadyobserved with only 5% C₄F₈ (Pc=117 mmHg).

[0046] After intra-aortic injection in rabbits (0.03 ml/kg), a slightprolongation of the contrast effect in the myocardium was noticedalready with 2% C₄F₈ (when compared to air). However with 5% C₄F₈, theduration of the contrast increased considerably as if above a thresholdvalue in the resistance to pressure, the persistence of the bubblesincreases tremendously (see FIG. 6). TABLE 2 Echogenicity Concen- airC₄F₈ Q Pc 1/(cm · sr) × tration Sample % vol % vol coeff. mmHg 100(bubbles/ml) A 100 0 1.0 43 1.6 1.8 × 10⁸ B 95 5 1.4 117 2.2 3.1 × 10⁸ C90 10 1.7 152 3.1 4.7 × 10⁸ D 75 25 3.3 197 3.5 4.9 × 10⁸ E 65 35 4.6209 3.4 4.3 × 10⁸ F 59 41 5.5 218 2.8 4.0 × 10⁸ G 0 100 1531 225 2.3 3.8× 10⁸

[0047] Here again, this combination of gases provided very good imagesat 5% of gas B in the mixture, while excellent images of the left heartwere obtained with the mixtures containing up to 25% of octafluorocyclobutane.

[0048] Corresponding diagram of critical pressure as a function of C₄F₈in the mixture with air is given in FIG. 2. This example again showsthat the use of mixture of gases allows to improve considerably theresistance to pressure of air bubbles simply by adding a smallpercentage of a high molecular weight/low solubility gas. The figurefurther shows that by appropriate selection of the gas mixture itbecomes possible to obtain any desired resistance to pressure.

EXAMPLE 3

[0049] The same lyophilisate as that described in Example 5 was used.The gas phase was made of dodecafluoropentane (C₅F₁₂) and air. C₅F₁₂ isa liquid at room temperature with a boiling point of 29.5° C. 24 mlglass vials each containing 50 mg of the PEG/DSPC/DPPG lyophilisateobtained as described in Example 5 were put under vacuum, closed undervacuum, then heated at 45° C. Small volumes (a few microliters) of C₅F₁₂were injected in the vials still at 45° C. through the stopper. Air wasthen introduced to restore atmospheric pressure in the vials. Aftercooling at room temperature, saline TABLE 3 half- life Inten air C₅F₁₂ QPc Echogen Conc. (t_(1/2)) Gray AUC Sample % vol % vol coeff. mmHg (cm ·sr)⁻¹ (bub/ml) sec level (t_(1/2)) A 100 0 1.0  43 0.017 1.8 × 10⁸ 11 2278 B 99.5 0.5 1.0  80* — — — — — C 98.6 1.4 1.1 133 0.026 3.9 × 10⁸ 1497 609 D 97.1 2.9 1.4 182 0.028 3.9 × 10⁸ 17 98 860 E 94.2 5.8 1.7 2950.040 5.2 × 10⁸ 59 99 3682 F 85.5 14.5 3.4 394 0.036 4.5 × 10⁸ 78 975141

[0050] (5 ml) was injected through the stopper and the vials werevigorously agitated. The actual percentage of C₅F₁₂ in the gas phase wascalculated assuming full vaporization of the liquid introduced. This isan overestimate as at this temperature part of the liquid will not be ingaseous state. As shown in FIG. 3 an increase in the resistance topressure could already be detected with only 0.5% C₅F₁₂ in air. At 1.4%C₅F₁₂ the resistance to pressure exceeded 130 mm Hg. These suspensionswere also injected intravenously into minipigs (0.5 ml per 15 kg).Intensity was measured by videodensitometry as described in Example 1.As shown in Table 3, maximum intensity was already obtained with 1.4%C₅F₁₂. Higher percentages of C₅F₁₂ result into prolongation of the halflife and increase in the AUC. The half life (t_(1/2)) was determined asthe time elapsed between injection and the time at which the intensityhad dropped to 50% of its maximum value. The area under the curve (AUC)was measured until t_(1/2).

[0051] The examples 1-3 also demonstrate that contrary to the statementsmade in WO-A-93/05819 it is possible to obtain outstanding contrastenhancing agents from gas mixtures whose Q values are smaller and incertain cases much smaller than 5.

EXAMPLE 4

[0052] Fifty eight milligrams of diarachidoylphosphatidylcholine (DAPC),2.4 mg of dipalmitoylphosphatidic acid (DPPA) both from Avanti PolarLipids (USA) and 3.94 g of polyethyleneglycol (PEG 4000 from Siegfried)were dissolved at 60° C. in tert-butanol (20 ml) in a round-bottom glassvessel. The clear solution was rapidly cooled at −45° C. andlyophilized. Aliquots (25 mg) of the white cake obtained were introducedin 10 ml glass vials.

[0053] Tedlar® gas sampling bags were filled with gases, one with airand one with sulfur hexafluoride (SF₆). Pre-determined volumes of thegases were collected from each bag through the septum by using twoseparate syringes and the contents mixed via a three way stopcock. Theresulting gas mixtures were introduced into 10 ml glass vials which wereevacuated and closed with rubber stopper while still under vacuum. Sevenvials contained gas mixtures of air and SF₆ in different proportions.The concentration of SF₆ was between 0 to 100%. The actual percentage ofSF₆ in the gas phase was confirmed by densimetry (A. Paar densimeter).Saline (0.9% NaCl) was then injected through the stopper into each vial(5 ml per vial) and the powder dissolved by vigorous shaking. Theresulting microbubble suspensions were evaluated in vitro and in vivo.The resistance to pressure P_(c) was determined using a nephelometricassay and the backscatter coefficient was measured using a pulse echoset up (both described in EP-A-0 554 213). The bubble concentration andmean bubble size were determined by analysis with a Coulter MultisizerII (Coulter Electronics Ltd). The results obtained were virtually thesame to those given for Example 1. TABLE 4 Gas B Pc Gas A Gas BSolubility* Solubility* Gas A Gas B % vol mmHg M_(wt) M_(wt) Gas A Gas BO₂ C₄F₈ 0 40 32 200 0.083 0.016 C₄F₈ 5 112 C₄F₈ 10 148 CO₂ C₄F₈ 0 50 44200 0.74 0.016 C₄F₈ 5 — C₄F₈ 10 204 CHClF₂ C₄F₈ 0 — 86.5 200 0.78 0.016C₄F₈ 5 106 C₄F₈ 10 163 Xenon C₄F₈ 0 50 131 200 0.108 0.016 C₄F₈ 5 147C₄F₈ 10 181 SF₆ C₄F₈ 0 124 146 200 0.005 0.016 C₄F₈ 5 159 C₄F₈ 10 193 N₂SF₆ 0 55 28 146 0.0144 0.005 SF₆ 5 80 SF₆ 10 108 CF₄ SF6 0 84 182 1460.0038 0.005 SF₆ 5 91 SF₆ 10 106 Xenon SF₆ 0 50 131 146 0.108 0.005 SF₆5 67 SF₆ 10 83

EXAMPLE 5

[0054] A PEG/DSPC/DPPG lyophilisate was prepared as described in Example4 using 30 mg of distearoylphosphatidylcholine (DSPC) and 30 mgdipalmitoyl-phosphatidylglycerol (DPPG) (both from SYGENA, Switzerland).Aliquots (25 mg) of the resulting cake were introduced in 10 ml glassvials. Different gas mixtures were introduced in various vials bywithdrawing appropriate volumes from Tedlar® bags filled with thevarious gases. Table 4 shows the gas mixtures investigated, theirmolecular weight and their solubilities (expressed as Bunsencoefficient) and the resistance to pressure of the microbubblesobtained. It is particularly interesting to note that highly solublegases such as CO₂, xenon, CHClF₂ which alone are very poor in theirability to form stable and resistant bubbles are nevertheless able togive rise to highly stable bubbles provided a small percentage of a gassuch as SF₆ or C₄F₈ is added.

EXAMPLE 6

[0055] The method of the invention was applied to a microbubblesuspension prepared as described in Example 1 of WO 92/11873. Threegrams of Pluronic® F68 (a copolymer or polyoxyethylene-polyoxypropylenewith a TABLE 5 Pc right ventr. opacif. left ventr. opacif. (mmHg)t_(1/2) intens AUC t_(1/2) intens AUC air C₄F₈ % vol % vol 100 0 54 4 96280 9 101 514  99 1 89 7 98 377 12 98 632  95 5 136 14 94 829 40 1012693 air C₅F₁₂  95 5 177 * * * 43 111 3249

[0056] molecular weight of 8400), 1 g of dipalmitoylphosphatidylglyceroland 3.6 g of glycerol were added to 80 ml of distilled water. Afterheating at about 80° C. a clear homogenous solution was obtained. Thetenside solution was cooled to room temperature and the volume adjustedto 100 ml. The bubble suspension was obtained by using two syringesconnected via a three-way valve. One of the syringes was filled with 5ml of the tenside solution while the other was filled with 0.5 ml of airor air/C₄F₈ mixture (see Table 5). The three way valve was filled withthe tenside solution before it was connected to the gas-containingsyringe. By alternatively operating the two pistons, the tensidesolution was transferred back and forth between the two syringes (5times in each direction) and milky suspensions were obtained. Afterdilution (1/50) in distilled water saturated with air the resistance topressure (Pc) was determined. Aliquots were injected intravenously intoanaesthethized rabbits (0.03 ml/kg) and echographic images of the leftventricle were recorded. The area under the curve (AUC) as well as thehalf life (t_(1/2)) were determined. A considerable increase of thehalf-life and AUC was observed when using 5% C₄F₈ (compared to air).Similar results were obtained with 5% C₅F₁₂.

EXAMPLE 7

[0057] A suspension of microbubbles was obtained as described inWO-A-93/05819 using mixtures of air and octafluorocyclobutane C₄F₈. Anaqueous solution containing sorbitol (20 g), NaCl (0.9 g), soybean oil(6 ml), Tween 20 TABLE 6 right left right left air C₄F₈ ventr. ventr.air C₅F₁₂ ventr. ventr. % vol % vol opacif. opacif. % vol % vol opacif.opacif. 100 0 + − 100 0 + − 99 1 + − 99 1 + + 95 5 ++ − 95 5 ++ ++

[0058] (0.5 ml) was prepared and adjusted to 100 ml of distilled water.10 ml of this solution was taken up in a 10 ml syringe. A second 10 mlsyringe was filled with mixtures of air and C₄F₈. The two syringes wereconnected via a three way stopcock. By operating alternatively each ofthe two pistons for a total of 20 times, milky suspensions wereobtained. These suspensions were tested for their resistance topressure. Aliquots were also injected intravenously into anaesthethizedrabbits (0.1 ml/kg) and echographic images of the left ventricle wererecorded. Interestingly no contrast was detected in the left ventriclewith 1% or even 5% C₄F₈. However, left ventricle opacification wasobtained with 1% and even more with 5% of C₅F₁₂.

EXAMPLE 8

[0059] A PEG/DSPC/DPPG lyophilisate was prepared as described in Example4 using 30 mg of distearoylphosphatidylcholine (DSPC) and 30 mgdipalmitoyl-phosphatidylglycerol (DPPG) (both from SYGENA, Switzerland).Aliquots (25 mg) of the resulting cake were introduced in 10 ml glassvials. Different gas mixtures were introduced in various vials bywithdrawing appropriate volumes from Tedlar® bags filled with thevarious gases. Table 7 shows the gas mixtures investigated and theresistance to pressure of the microbubbles obtained. It is noteworthythe high molecular weight gas may even be a mixture of two or more gaseswith high molecular weight and TABLE 7 C₄F₈ CF₄ air Pc Sample % vol %vol % vol mmHg Absorbance A₁ 5 15 80 113 0.284 A₂ 10 10 80 147 0.281 A₃15 5 80 167 0.281

[0060] solubility (expressed as Bunsen coefficient) which is below0.0283. It follows that in place of a single gas (B), mixtures of two ormore activating or minor component gases may also be used. Although, inthis example, the critical pressure is proportional to the percentage ofthe heavier of the two components, it is believed that othercombinations of gases may further lower the total amount of theinsoluble gas(es) in the mixture through synergy.

1-24. (cancelled)
 25. A method of making an ultrasound contrast agentcomprising suspending gas filled microbubbles in a physiologicallyacceptable aqueous carrier comprising film forming saturatedphospholipid surfactants present in laminar and/or lamellar form, inwhich the gas filling the microbubbles is a mixture of at least twobiocompatible gases A and B, wherein A is selected from the groupconsisting of air, oxygen, nitrogen and carbon dioxide, and B is C₄F₁₀.26. The method of claim 25, wherein A is air.
 27. The method of claim25, wherein A is nitrogen.
 28. The method of claim 27, wherein thesaturated phospholipid surfactants are selected from the groupconsisting of phosphatidic acid, phosphatidylcholine,phosphatidylethanolamine, phosphatidylserine, phosphatidylglycerol,phosphatidyl linositol, cardiolipin, sphingomyelin and mixtures thereof.29. The method of claim 27, wherein the saturated phospholipidsurfactants comprise phosphatidic acid and phosphatidylcholine.